SUMMARY Acute myeloid leukemia (AML) is the most common adult acute leukemia. Although frontline treatment of AML with cytotoxic chemotherapy achieves high remission rates, 75-80% of patients will either not respond or will relapse after initial therapy, and most patients will die of their disease within 2 years. Thus, there is a great need for more effective and less toxic therapies for AML. The goal of this proposal is to develop novel therapeutic strategies to treat AML based on the unique metabolic dependencies of these leukemias, particularly those dependencies that arise after treatment with inhibitors targeting activated tyrosine kinases (TK) like FLT3. We have shown that inhibition of FLT3 in AML cells results in dramatic metabolic changes and novel mitochondrial dependencies. MCJ is a mitochondrial protein that interacts with Complex I of the mitochondrial electron transport chain (ETC) and attenuates Complex I activity. MCJ expression is lost in a number of cancers and we now show that MCJ expression is lost in multiple FLT3 activated AML cell lines. Our collaborators have generated MCJ agonistic peptides that can restore MCJ activity in cancer cells lacking MCJ, improving therapeutic responses. Importantly, we demonstrate that peptide-mediated reactivation of MCJ results in synergistic elimination of FLT3-dependent AML cell line when combined with FLT3 TK inhibitors (TKI) in vitro. We hypothesize that restoration of MCJ activity in a subset of AMLs using MCJ agonistic peptides will disrupt ETC supercomplexes, promote loss of mitochondrial transmembrane potential (?m), reduce respiration, increase production of mitochondrial ROS, and promote apoptosis. Our goal is to demonstrate that MCJ agonistic peptides synergize with FLT3 inhibitors in the treatment of AML, with the goal of developing therapies to improve outcomes for AML patients. In this application we will examine: 1) the mechanism underlying MCJ agonist- induced apoptosis in AML cells, using AML cell lines and primary cells in vitro, and 2) the efficacy of MCJ agonists in the treatment of AML in vivo in combination with FLT3 inhibition using mouse models and primary human AML cells.